DESCRIPTION (Adapted from abstract): X-ray crystallography will be used to study the structures of replicases (polymerase, sliding clamp protein, and complex that loads clamp onto DNA). Completed work has shown that the processivity factors from bacteria (beta subunit), eukaryotes (PCNA), and bacteriophage T4 (gene 45 protein) are ring-shaped proteins that share a commo architecture. These provide a sliding platform to which the polymerase machinery is tethered. Each of these replicases contains a multi-subunit clamp-loader. An active clamp-loader complex from E. coli (gamma, delta, and delta prime subunits) has been crystallized. An alternative strategy is to use proteins from thermophilic organisms. Several subunits of the replicase of the eubacterium Thermus thermophilis (T. th.) have been cloned. The DNA-dependent ATPase subunit of the clamp-loader of T. th. (gamma subunit) has been crystallized. The structure of the DNA polymerase/exonuclease from an extreme archaebacterial thermophile Desulfurucoccus tok, which is homologous to the PCNA-dependent mammalian DNA polymerases delta and epsilon, will be determined from crystals that diffract to 2.4 A. Although DNA replicases from various organisms share a common mechanistic framework, the protein components are quite divergent in sequence. The structural information resulting from this work may set the stage for structure-based design of replicase inhibitors with potential for high specificity.